PROJECT SUMMARY/ABSTRACT Exposure of cells to environmental agents, such as radiation, heavy metals, air pollutants and mutagenic chemicals, generates DNA double-strand breaks (DSB)s and other chromosomal lesions, and can also cause replicative stress. Such environmentally induced chromosomal lesions and stress are eliminated by a conserved mechanism - homologous recombination (HR). Defects in HR and its deregulation lead to genome destabilization, cancer, and other diseases. Several nucleic acid motor proteins, including helicases, have been implicated in mechanisms that affect HR outcome and DNA damage repair, relieve cells from replication fork stress, and mediate the resolution of R-loops. ZGRF1, a nucleic acid motor, has been implicated in HR and DNA crosslink repair in siRNA- based genomic screens. We have found that engineered ZGRF1-/- cells are hypersensitivity to mitomycin C (MMC) treatment and accumulate chromosome aberrations upon drug treatment. Importantly, purified ZGRF1 shows DNA dependent ATPase, D-loop and R-loop dissociation, and replication fork regression activities, suggesting that ZGRF1 functions directly to regulate HR and mediate replication fork repair and R-loop resolution. In this project, we will apply our considerable expertise in molecular studies of nucleic acid motor proteins to define the mechanisms by which ZGRF1 accomplishes its biological functions. In Aim 1, we will perform a variety of genetic and cytological studies to examine ZGRF1 mutant cells for defects in DNA damage repair, replication fork maintenance/repair, and also R-loop resolution. We will ascertain the biological relevance of the ZGRF1 nucleic acid motor activity with an ATPase defective mutant that we have generated. In Aim 2, we will define the biochemical attributes of ZGRF1, and carry out co- immunoprecipitation and biochemical pulldown to identify interactors of this motor protein. In Aim 3, we will investigate the biochemical and genetic defects of ZGRF1 mutants altered in the zinc finger domain, impaired for protein-protein interactions, or found in cancer. The results from our project will shed light on the roles of ZGRF1 in nuclear processes that are germane for delineating how disease causative mutations and chromosome rearrangements arise in cells deficient in nucleic acid motors. The results on R-loop resolution are expected to contribute toward the development of novel strategies to avoid the accumulation of R-loops upon exposure to environmental stress and mutagens.